Low-dose hydralazine prevents fibrosis in a murine model of acute kidney injury–to–chronic kidney disease progression
2016; Elsevier BV; Volume: 91; Issue: 1 Linguagem: Inglês
10.1016/j.kint.2016.07.042
ISSN1523-1755
AutoresBjörn Tampe, Ulrike Steinle, Désirée Tampe, Julienne L. Carstens, Peter Korsten, Elisabeth M. Zeisberg, Gerhard A. Müller, Raghu Kalluri, Michael Zeisberg,
Tópico(s)Acute Kidney Injury Research
ResumoAcute kidney injury (AKI) and progressive chronic kidney disease (CKD) are intrinsically tied syndromes. In this regard, the acutely injured kidney often does not achieve its full regenerative capacity and AKI directly transitions into progressive CKD associated with tubulointerstitial fibrosis. Underlying mechanisms of such AKI-to-CKD progression are still incompletely understood and specific therapeutic interventions are still elusive. Because epigenetic modifications play a role in maintaining tissue fibrosis, we used a murine model of ischemia-reperfusion injury to determine whether aberrant promoter methylation of RASAL1 contributes causally to the switch between physiological regeneration and tubulointerstitial fibrogenesis, a hallmark of AKI-to-CKD progression. It is known that the antihypertensive drug hydralazine has demethylating activity, and that its optimum demethylating activity occurs at concentrations below blood pressure–lowering doses. Administration of low-dose hydralazine effectively induced expression of hydroxylase TET3, which catalyzed RASAL1 hydroxymethylation and subsequent RASAL1 promoter demethylation. Hydralazine-induced CpG promoter demethylation subsequently attenuated renal fibrosis and preserved excretory renal function independent of its blood pressure–lowering effects. In comparison, RASAL1 demethylation and inhibition of tubulointerstitial fibrosis was not detected upon administration of the angiotensin-converting enzyme inhibitor Ramipril in this model. Thus, RASAL1 promoter methylation and subsequent transcriptional RASAL1 suppression plays a causal role in AKI-to-CKD progression. Acute kidney injury (AKI) and progressive chronic kidney disease (CKD) are intrinsically tied syndromes. In this regard, the acutely injured kidney often does not achieve its full regenerative capacity and AKI directly transitions into progressive CKD associated with tubulointerstitial fibrosis. Underlying mechanisms of such AKI-to-CKD progression are still incompletely understood and specific therapeutic interventions are still elusive. Because epigenetic modifications play a role in maintaining tissue fibrosis, we used a murine model of ischemia-reperfusion injury to determine whether aberrant promoter methylation of RASAL1 contributes causally to the switch between physiological regeneration and tubulointerstitial fibrogenesis, a hallmark of AKI-to-CKD progression. It is known that the antihypertensive drug hydralazine has demethylating activity, and that its optimum demethylating activity occurs at concentrations below blood pressure–lowering doses. Administration of low-dose hydralazine effectively induced expression of hydroxylase TET3, which catalyzed RASAL1 hydroxymethylation and subsequent RASAL1 promoter demethylation. Hydralazine-induced CpG promoter demethylation subsequently attenuated renal fibrosis and preserved excretory renal function independent of its blood pressure–lowering effects. In comparison, RASAL1 demethylation and inhibition of tubulointerstitial fibrosis was not detected upon administration of the angiotensin-converting enzyme inhibitor Ramipril in this model. Thus, RASAL1 promoter methylation and subsequent transcriptional RASAL1 suppression plays a causal role in AKI-to-CKD progression. Acute kidney injury (AKI) and chronic progressive kidney disease (CKD) are principal problems in nephrology.1Venkatachalam M.A. Griffin K.A. Lan R. et al.Acute kidney injury: a springboard for progression in chronic kidney disease.Am J Physiol Renal Physiol. 2010; 298: F1078-F1094Crossref PubMed Scopus (391) Google Scholar, 2Thadhani R. Pascual M. Bonventre J.V. Acute renal failure.N Engl J Med. 1996; 334: 1448-1460Crossref PubMed Scopus (1511) Google Scholar, 3Chawla L.S. Eggers P.W. Star R.A. 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Zeisberg E.M. et al.Methylation determines fibroblast activation and fibrogenesis in the kidney.Nat Med. 2010; 16: 544-550Crossref PubMed Scopus (487) Google Scholar We further showed that RASAL1 methylation and subsequent transcriptional silencing of RASAL1 causally contributes to sustained fibroblast activation (by increasing intrinsic Ras-guanosine triphosphate levels, similar to cancer cells).11Bechtel W. McGoohan S. Zeisberg E.M. et al.Methylation determines fibroblast activation and fibrogenesis in the kidney.Nat Med. 2010; 16: 544-550Crossref PubMed Scopus (487) Google Scholar We further discovered that RASAL1 is hypermethylated consistently in murine models of chronic progressive fibrosis (including unilateral ureteral obstruction, subtotal nephrectomy, and nephrotoxic serum nephritis),28Tampe B. Tampe D. Zeisberg E.M. et al.Induction of Tet3-dependent epigenetic remodeling by low-dose hydralazine attenuates progression of chronic kidney disease.EBioMedicine. 2015; 2: 19-36Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 29Tampe B. Tampe D. Muller C.A. et al.Tet3-mediated hydroxymethylation of epigenetically silenced genes contributes to bone morphogenic protein 7-induced reversal of kidney fibrosis.J Am Soc Nephrol. 2014; 25: 905-912Crossref PubMed Scopus (94) Google Scholar but not in a murine model of fully reversible ischemia-reperfusion injury.11Bechtel W. McGoohan S. Zeisberg E.M. et al.Methylation determines fibroblast activation and fibrogenesis in the kidney.Nat Med. 2010; 16: 544-550Crossref PubMed Scopus (487) Google Scholar Furthermore, we discovered that therapeutic RASAL1 demethylation in vivo could be achieved either by administration of the prototypical demethylating drug 5’-azacytidine (which is in clinical use for treatment of refractory myelodysplastic syndrome despite its substantial side effects),11Bechtel W. McGoohan S. Zeisberg E.M. et al.Methylation determines fibroblast activation and fibrogenesis in the kidney.Nat Med. 2010; 16: 544-550Crossref PubMed Scopus (487) Google Scholar but also by administration of hydralazine (and its derivate dihydralazine), and such RASAL1 demethylation correlated with ameliorated fibrosis.28Tampe B. Tampe D. Zeisberg E.M. et al.Induction of Tet3-dependent epigenetic remodeling by low-dose hydralazine attenuates progression of chronic kidney disease.EBioMedicine. 2015; 2: 19-36Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar Hydralazine was first approved by the U.S. Food and Drug Administration as an anti-hypertensive drug in 1952.30Zeisberg M. Zeisberg E.M. Precision renal medicine: a roadmap towards targeted kidney fibrosis therapies.Fibrogenesis Tissue Repair. 2015; 8: 16Crossref PubMed Scopus (12) Google Scholar Today, it has its place in clinical practice as a third-line antihypertensive regimen in patients with complicated hypertension, as an antihypertensive in intensive care unit settings, as a second-line therapeutic for chronic heart failure (when use of inhibitors of the renin-angiotensin-aldosterone system [RAAS] is not warranted), or because of its safety profile in pregnancy hypertension.30Zeisberg M. Zeisberg E.M. 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Zeisberg E.M. et al.Induction of Tet3-dependent epigenetic remodeling by low-dose hydralazine attenuates progression of chronic kidney disease.EBioMedicine. 2015; 2: 19-36Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar Here, we aimed to explore if aberrant promoter methylation of RASAL1 contributes causally to the shift from regeneration to fibrosis that is associated with AKI-to-CKD progression upon acute kidney injury, and, if so, whether such fibrotic response could be prevented by administration of low-dose hydralazine. We show that fibrogenesis ensuing after severe ischemia-reperfusion injury (IRI) is associated with RASAL1 promoter methylation and transcriptional silencing, whereas moderate IRI followed by full recovery is not.38Yang L. Besschetnova T.Y. Brooks C.R. et al.Epithelial cell cycle arrest in G2/M mediates kidney fibrosis after injury.Nat Med. 2010; 16 (1p following 143): 535-543Crossref PubMed Scopus (906) Google Scholar, 39Le Clef N. Verhulst A. D'Haese P.C. Vervaet B.A. unilateral renal ischemia-reperfusion as a robust model for acute to chronic kidney injury in mice.PLoS One. 2016; 11: e0152153Crossref Scopus (103) Google Scholar We provide evidence that rescue of RASAL1 expression in RASAL1-transgenic mice inhibits renal fibrogenesis upon severe IRI, supporting that loss of RASAL1 causally contributes to AKI-to-CKD progression. We further show that administration of low-dose hydralazine during acute kidney injury at a dose of 5 mg/kg normalizes aberrant RASAL1 promoter methylation and effectively prevents AKI-to-CKD progression and renal fibrogenesis upon severe IRI. In contrast, administration of the angiotensin-converting enzyme inhibitor (ACEi) ramipril during acute kidney injury had no demethylating activity on renal fibroblasts and no beneficial effects on fibrogenesis or AKI-to-CKD progression in this model. Previously, we established that Rasal1 promoter methylation contributed causally to the progression of tubulointerstitial fibrosis in multiple murine models of chronic kidney disease (unilateral ureteral obstruction, folic acid nephropathy, and nephrotoxic serum nephritis),11Bechtel W. McGoohan S. Zeisberg E.M. et al.Methylation determines fibroblast activation and fibrogenesis in the kidney.Nat Med. 2010; 16: 544-550Crossref PubMed Scopus (487) Google Scholar, 28Tampe B. Tampe D. Zeisberg E.M. et al.Induction of Tet3-dependent epigenetic remodeling by low-dose hydralazine attenuates progression of chronic kidney disease.EBioMedicine. 2015; 2: 19-36Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 29Tampe B. Tampe D. Muller C.A. et al.Tet3-mediated hydroxymethylation of epigenetically silenced genes contributes to bone morphogenic protein 7-induced reversal of kidney fibrosis.J Am Soc Nephrol. 2014; 25: 905-912Crossref PubMed Scopus (94) Google Scholar whereas Rasal1 was not methylated in a rodent model of fully reversible ischemia-reperfusion injury.11Bechtel W. McGoohan S. Zeisberg E.M. et al.Methylation determines fibroblast activation and fibrogenesis in the kidney.Nat Med. 2010; 16: 544-550Crossref PubMed Scopus (487) Google Scholar Based on these findings, we hypothesized that Rasal1 promoter methylation could be causally involved in switching AKI recovery to AKI-to-CKD fates. To test this hypothesis, we aimed to use models allowing for direct comparison of involved pathomechanisms owing to comparable insults and injury modes. For this purpose, we decided to use 2 mouse models of ischemia-reperfusion injury: a rodent model of IRI with effective recovery from injury (moderate IRI) and a mouse model leading to tubulointerstitial fibrosis (severe IRI) after 42 days (Figure 1a–g).38Yang L. Besschetnova T.Y. Brooks C.R. et al.Epithelial cell cycle arrest in G2/M mediates kidney fibrosis after injury.Nat Med. 2010; 16 (1p following 143): 535-543Crossref PubMed Scopus (906) Google Scholar, 39Le Clef N. Verhulst A. D'Haese P.C. Vervaet B.A. unilateral renal ischemia-reperfusion as a robust model for acute to chronic kidney injury in mice.PLoS One. 2016; 11: e0152153Crossref Scopus (103) Google Scholar, 40Delbridge M.S. Shrestha B.M. Raftery A.T. et al.The effect of body temperature in a rat model of renal ischemia-reperfusion injury.Transplant Proc. 2007; 39: 2983-2985Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Such severe IRI was associated with impaired recovery from tubular injury (Figure 1a and b), increase of the relative interstitial volume (Figure 1a and c), accelerated deposition of type I collagen (Collagen-1) (Figure 1a and d), accumulation of α-smooth muscle actin (α-SMA)-positive myofibroblasts (Figure 1a and e), increased intrarenal mRNA expression levels of Collagen-1a1 (Figure 1f) and Acta2 (encoding α-SMA) (Figure 1g), and accumulation of proliferating Ki67-positive interstitial cells (Figure 1a and h) at 42 days after renal injury. In the moderate IRI model correlating with completed tubular regeneration, proliferative activity of tubular epithelial cells (as assessed by immunolabeling with proliferation marker Ki67) had ceased after 42 days, correlating with completed tubular regeneration (Figure 1a and i). Incomplete tubular regeneration after 42 days in the severe IRI model correlated with sustained Ki67 immunolabeling of tubular epithelial cells, indicating ongoing response to injury and prolonged repair (Figure 1a and i), in line with previous findings.38Yang L. Besschetnova T.Y. Brooks C.R. et al.Epithelial cell cycle arrest in G2/M mediates kidney fibrosis after injury.Nat Med. 2010; 16 (1p following 143): 535-543Crossref PubMed Scopus (906) Google Scholar, 41Rodriguez-Romo R. Benitez K. Barrera-Chimal J. et al.AT1 receptor antagonism before ischemia prevents the transition of acute kidney injury to chronic kidney disease.Kidney Int. 2016; 89: 363-373Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar AKI-to-CKD progression with tubulointerstitial fibrogenesis upon severe IRI was associated with increased Rasal1 promoter methylation (Figure 1j and k) and consecutive loss of intrarenal Rasal1 mRNA expression levels (Figure 1l), whereas Rasal1 methylation and transcriptional suppression was not observed upon moderate IRI associated with full regeneration (Figure 1j–l). Loss of intrarenal Rasal1 during AKI-to-CKD progression was confirmed, especially in interstitial compartments (Figure 1m and n), in line with previous findings that loss of Rasal1 is involved in determining fibroblast activation in the kidney.11Bechtel W. McGoohan S. Zeisberg E.M. et al.Methylation determines fibroblast activation and fibrogenesis in the kidney.Nat Med. 2010; 16: 544-550Crossref PubMed Scopus (487) Google Scholar In summary, tubulointerstitial fibrosis in response to severe IRI was associated with Rasal1 promoter methylation, whereas Rasal1 methylation was not observed when kidneys were capable of regenerating upon moderate IRI, suggesting that Rasal1 promoter methylation contributes causally to AKI-to-CKD progression. To further substantiate the mechanistic link between aberrant Rasal1 promoter methylation and determination of AKI-to-CKD progression, we next challenged transgenic mice harboring transgenes for doxycycline-inducible RASAL1 overexpression (rtTAhCMV;hRASAL1-pTreTight, referred to as hRASAL1oe) with severe IRI. As described previously,28Tampe B. Tampe D. Zeisberg E.M. et al.Induction of Tet3-dependent epigenetic remodeling by low-dose hydralazine attenuates progression of chronic kidney disease.EBioMedicine. 2015; 2: 19-36Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar these mice show robust RASAL1 overexpression upon administration of doxycycline, and transgene expression is under control of a cytomegalovirus minimal promoter that is devoid of CpG islands and hence resistant to potential transcriptional silencing through promoter methylation during AKI-to-CKD progression (Figure 2a and b). When challenged with severe IRI, kidneys of wild-type control mice receiving doxycycline showed interstitial fibrosis (Figure 2c–e) and fibroblast accumulation (Figure 2c,f,g). Transgenic RASAL1 overexpression in rtTAhCMV;hRASAL1-pTreTight mice upon doxycycline administration (hRASAL1oe) attenuated AKI-to-CKD progression and kidney fibrogenesis (Figure 2c–e). Reduced fibrosis correlated with attenuated fibroblast accumulation (Figure 2c,f,g) and improved renal histology (microvascular rarefaction and intrarenal inflammation) (Supplementary Figure S1 online),1Venkatachalam M.A. Griffin K.A. Lan R. et al.Acute kidney injury: a springboard for progression in chronic kidney disease.Am J Physiol Renal Physiol. 2010; 298: F1078-F1094Crossref PubMed Scopus (391) Google Scholar, 42Bonventre J.V. Primary proximal tubule injury leads to epithelial cell cycle arrest, fibrosis, vascular rarefaction, and glomerulosclerosis.Kidney Int Suppl. 2011; 2014: 39-44Google Scholar, 43Mazzali M. Jefferson J.A. Ni Z. et al.Microvascular and tubulointerstitial injury associated with chronic hypoxia-induced hypertension.Kidney Int. 2003; 63: 2088-2093Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 44Lin C.H. Chen J. Zhang Z. et al.Endostatin and transglutaminase 2 are involved in fibrosis of the aging kidney.Kidney Int. 2016; 89: 1281-1292Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar showing that fibroblast activation is attenuated and kidneys are protected from AKI-to-CKD progression when Rasal1 expression was rescued. In summary, our studies showed that interstitial fibrosis upon severe ischemia-reperfusion injury was associated with loss of intrarenal Rasal1 expression, and restoration of intrarenal Rasal1 expression levels effectively protects from AKI-to-CKD progression. Because our data showed a contribution of Rasal1 promoter methylation to AKI-to-CKD progression (compare with Figure 1), restoration of intrarenal Rasal1 protects from AKI-to-CKD progression (compare with Figure 2), and we previously established the activity of low-dose hydralazine to demethylate Rasal1 in murine models of kidney fibrosis,28Tampe B. Tampe D. Zeisberg E.M. et al.Induction of Tet3-dependent epigenetic remodeling by low-dose hydralazine attenuates progression of chronic kidney disease.EBioMedicine. 2015; 2: 19-36Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar we next aimed to explore the possibility that administration of hydralazine could attenuate AKI-to-CKD progression and renal fibrogenesis upon severe IRI by normalization of aberrant Rasal1 promoter methylation. To exclude the possibility that potential renoprotective effects upon hydralazine administration were caused by blood pressure–lowering effects, we administered hydralazine at a dosage of 5 mg/kg per day, established to exert optimum demethylat
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